DESCRIPTION: The long-term goals of this proposal are to develop and demonstrate synthetic strategies necessary for the synthesis and utilization of specifically modified or labeled RNA fragments. Molecules that are site-specifically labeled with 15N and 13C can provide unique and model-independent insight into local RNA structure, including hydrogen bonding, protonation, hydration, protection from hydration and stacking. The utility of 15N specifically labeled nucleosides will be expanded by introducing 13C into the C8 position of both purines and the C2 position of adenine. The resulting coupling with 15N in only one of a pair of purines will allow otherwise similar NMR resonances to be distinguished. Furthermore, the 13C can provide independent information on base stacking. The procedures developed for RNA synthesis using specific 15N and 13C labels will be made more widely available by adapting them to the phosphoramidite method. Since many of the simpler RNA motifs like non-canonical base pairs, single bulges, and tetraloops have now been characterized by 15N NMR, the new NMR work will focus primarily on more complex systems, such as internal loops, large hairpin loops, pseudoknots, kissing loops, tetraloop receptors, ribozymes, and protein-RNA complexes. The use of 15N NMR to define metal ion binding to RNA, a crucial aspect of its structure and function, will be explored.